The Structural Basis of Actin Organization by Vinculin and Metavinculin

Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5-Å-resolution cryo-electron microscopy reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal "strap" and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1' helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform that suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner, providing a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies

Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells

Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner.

The spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. Each mammalian kinetochore binds many microtubules, but how many attached microtubules are required to turn off the checkpoint, and how the kinetochore monitors microtubule numbers, are not known and are central to understanding SAC mechanisms and function. To address these questions, here we systematically tune and fix the fraction of Hec1 molecules capable of microtubule binding. We show that Hec1 molecules independently bind microtubules within single kinetochores, but that the kinetochore does not independently process attachment information from different molecules. Few attached microtubules (20% occupancy) can trigger complete Mad1 loss, and Mad1 loss is slower in this case. Finally, we show using laser ablation that individual kinetochores detect changes in microtubule binding, not in spindle forces that accompany attachment. Thus, the mammalian kinetochore responds specifically to the binding of each microtubule and counts microtubules as a single unit in a sensitive and switch-like manner. This may allow kinetochores to rapidly react to early attachments and maintain a robust SAC response despite dynamic microtubule numbers

Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament (“F-actin”) assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, “jordan”). Here, we present cryo–electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. Adenosine diphosphate–bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide state–dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height

Structure and dynamics of single-isoform recombinant Neuronal Human Tubulin

Microtubules are polymers that cycle stochastically between polymerization and depolymerization i.e., they exhibit 'dynamic instability'. This behavior is crucial for cell division, motility and differentiation. While studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogenous, isotypically pure, engineered tubulin. Here we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2 Å structure of unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but is distinguished by subtle differences at polymerization interfaces, which are hotspots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogenous microtubules undergo dynamic instability but they polymerize slower and catastrophe less frequently. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient, but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogenous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well-defined tubulin species and is a first and necessary step towards uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics

Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome

Quantitative measurement of the number of Cse4, CBF3, and Ndc80 proteins at kinetochores reveals a 2.5–3-fold increased copy number relative to prior estimates

The structure of the complex between a-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in a-tubulin–b-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and a-tubulin (aEB) complex, which is formed upon a-tubulin–b-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the aEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the a-tubulin–b-tubulin interface that is caused by a steric interaction between b-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the aEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating a-tubulin degradation